As already mentioned, the subject invention concerns determination of analyte concentrations in anticoagulated plasma by measurements on mixtures of blood and liquid reagent. The results of such determinations are needed to make medical diagnosis and to monitor the effects of medical treatments.
Determinations of analyte concentrations for medical purposes are traditionally performed at laboratories, distant from the patient. The results of the determinations are often needed at care facilities, close to the patient. This spatial situation creates a drive to perform analyte concentration determinations near the patient. Only anticoagulated blood can be transported from care facilities to laboratories. At laboratories, the analyte determinations are performed on the anticoagulated blood or on anticoagulated plasma prepared from the anticoagulated blood. Since anticoagulated plasma is easier to work with and easier to store, laboratory determinations of analyte concentrations are performed on anticoagulated plasma, to the extent possible. At the near-patient facilities the situation is different. Blood is readily available, but anticoagulated plasma is inconvenient or impossible to prepare. This gives rise to a situation where laboratory determinations of analyte concentrations are performed on anticoagulated plasma and near-patient determinations are performed on blood. The situation is unsatisfactory because rational medical practice requires the association between one analyte concentration value and one given patient at one given time. Given the choice, clinicians would prefer the values of analyte concentration in anticoagulated plasma, because these values are association with a greater wealth of clinical reference data. Apart from the nature of the sample, the subject invention concerns accuracy and reliability of analyte concentration determinations. It is recognized, that accuracy and reliability are fundamental for the medical usefulness of analyte concentration determinations.
Per definition, an analytical method is accurate if it generates results that are in agreement with those of a reference method. This also applies to near-patient methods to determine analyte concentrations. A rational design strategy to obtain an accurate near-patient method is to adopt the chemistry and the assay conditions of a reference method, or of a method proven to be accurate according to the above. This straightforward strategy is difficult to follow. Reference methods are laboratory methods that represent culminations of long-term collaborative researchers' efforts in laboratory milieus. These milieus and milieus of clinical laboratories are relatively similar. Assay condition that can be accomplished in one of these milieus can be accomplished in the other. The milieus within which near-patient concentration determinations are performed are markedly different. Already the first procedural step of a typical laboratory method, to mix a precise volume of anticoagulated plasma and a precise volume of a reagent, represents a near insurmountable hurdle at near-patient assay sites. At surgical theaters, primary care centers, doctors' offices and patients' homes, anticoagulated plasma is inconvenient to prepare, and precise volumes are difficult to accomplish. Therefore, the first procedural step of a near-patient method is typically; to mix an imprecisely defined volume of blood with a dry reagent. The designers of near-patient assay methods have not purposefully deviate from the assay conditions of the accurate laboratory methods; it has been forced upon them. Still, the deviations from laboratory assay conditions have inflicted accuracy flaws in the near-patient assay methods. This has caused concern and insecurity, and has compromised the safety and efficacy of medical diagnosis and treatments. A prudent strategy to improve the accuracy of near-patient analyte concentration determinations is therefore, 1) to identify the aspects of assay conditions of laboratory methods that promote accuracy, and 2) to persistently adhere to the identified aspects in design of near patient methods.
As stated above, reference methods, and accurate laboratory methods, are often wet-chemistry methods. A main reason for the success of the wet-chemistry methods is their universal potency in combating matrix effects. Mixing a small volume of sample with a large volume of reagent dilutes the sample. This diminishes all effects of the sample and sets the scene for assay conditions that selectively favor the effects of the analyte. The effects of the non-analytes of the sample, the matrix effects, are thereby disfavored and the accuracy of the assay enhanced.
Quantitative determination of analyte concentrations by wet-chemistry methods requires precise allotment of intended volumes, precisely what is difficult to accomplish at near-patient assay sites. The allotment may be well be precise, but in void of systems for checking volumetric equipment, properly trained laboratory personnel and other aspects of a well managed laboratory, the allotments of volumes at near-patient sites are bound to be inaccurate, e.g. differ unacceptably from was intended. Prior art approaches to solving the problem is to invent ‘user friendly’, inexpensive, precise and accurate volumetric devices with which near-patient methods may be practiced. Such approaches have experienced limited success.
Apart from the classical near-patient assay site mentioned above, near-patient assays are also performed at smaller laboratories and at divisions of larger laboratories. All near-patient assay sites share the aversion of preparing anticoagulated plasma but display a difference in their ability to accomplish precisely defined volumes. In the following, a distinction is made between near-patient assay sites and smaller laboratories. They share a preference for blood but differ in their ability to precisely allot intended volumes of blood and reagents.
Smaller laboratories and near-patient assay sites also share concerns regarding the reliability of analyte concentration determinations. Larger laboratories set the reliability standard. At larger laboratories, thousands of analyte concentrations per year, of a given kind, are performed. Around the clock, stationary, automated, reliable measurement and determination devices allot intended volumes of anticoagulated plasma and reagent, perform measurements and determine analyte concentrations. Control samples with assigned analyte concentration values are analysed regularly and the activities are supervised by specialized, well-trained technicians. The measurement and determination devices undergo periodic maintenance. Calibration, involving the whole procedural set up, is performed as required, particularly whenever procedural changes are made, e.g. when a new lot of reagent is introduced. Because of all this, a high level of assay reliability is reached at larger laboratories. It is no easy task to attain comparable assay reliability at smaller laboratories and at near-patient assay sites. A strategy for reliability improvement includes identifying reliability-enhancing routines practiced at larger laboratories, but not at the smaller laboratories and near-patient assay sites, and finding ways to make these routines or equivalents possible to practice also at the latter sites. Periodic maintenance of measuring and determination devices is one such measure. Calibration of the whole procedural set-up, upon introduction of procedural changes, is another. Regular analysis of control samples is a third.
For reasons of tradition, and to speed reactions, laboratory methods are typically performed at 37° C. With regard to accuracy, the temperature, it itself, is typical not crucial. If advantages in design of near-patient assay methods can be gained by performing the assay at measured ambient room temperature and accommodate the measurement in the determination, this should be considered. The reason is that measuring a temperature is much less demanding that keeping it at a defined level. The demand for 37° C. in near-patient methods is a likely source of imprecision and inaccuracy. At smaller laboratories and near-patient sites, analyte concentration determinations, of a given kind, are performed sporadically. Because of this, the measurement devices are not in constant operation. The demand for 37° C. requires temperature equilibration of measurement devices and reagents in immediate connection with the determination. This, apart from consuming valuable time, becomes a source of error. Since time is precious, the equilibration time will always be at a minimum, and always be somewhat insufficient. The somewhat insufficient temperature equilibration time will result in imprecise temperature definition, and cause assay imprecision. The somewhat insufficient temperature equilibration will also tend to give lower temperatures than the intended and cause assay inaccuracy. Assay time, imprecision and inaccuracy are reduced if temperature equilibration were avoided. Furthermore, assigning thermostat-heating blocks to oblivion, obviously, reduces the complexity and cost of measurement devices and markedly reduces their power consumption. This, in itself, may open the way to disposable or semi-disposable, light weight, portable, manufacturer calibrated and maintained assay equipment, which may increase accuracy and reliability of near-patient analyte concentration determinations, and reduce costs.
Further description of the background of the invention is by example, the determination of prothrombin time (PT).
According to prior art, there are two methods of PT determination. One is described in Quick A. The prothrombin time in hemophilia and obstructive jaundice. Journal of Biological Chemistry 1935; 109:73-74. The other is described in Owren P. Thrombotest. A new method for controlling anticoagulant therapy. Lancet 1959; ii: 754-758. Both methods are based on coagulation induced by cell membrane bound tissue factor. Hence, the reagent of both methods contains thromboplastin. However, there is an important difference. Apart from various salts and excipients, a Quick PT reagent contains only thromboplastin, whereas an Owren PT reagent also contains plasma depleted of proteins that bind to BaSO4. In particular, the depleted plasma is depleted of coagulation factors II, VII and X, but not depleted in two other protein components necessary for coagulation, coagulation factor V and fibrinogen. The Quick PT method relies on the sample, as a source of fibrinogen and coagulation factor V, and is profoundly affect by deficiencies and abnormalities of these. The Owren PT method is thus more specific for the factors of interest. Since coagulation factors II, VII and X, but not coagulation factor V and fibrinogen, are influenced by medical treatments with vitamin K antagonists, the Owren PT method is more specific to the effects of such treatments. The treatments are highly effective in preventing thrombosis and other coagulopathies and PT assays firmly established in monitoring these treatments to assure their safety and efficacy.
Fibrinogen is crucial in PT assays. It is the substance that forms the clot. No fibrinogen means no clot, no clotting time and no PT assay. If, the fibrinogen level falls below about 0.1 g/L in the mixture of sample and reagent, clot formation is severely hampered and the clotting end-point becomes dubious. Since plasma levels of fibrinogen range down to 1 g/l, plasma to reagent ratios below 1:10 are prohibited in the Quick PT method. No such limit exists for the Owren PT method, since the reagent contains fibrinogen, and the plasma to reagent ratio can be reduced much further than 1:10.
The Quick PT method specifies a reaction mixture composed one volume of anticoagulated plasma and two volumes of reagent. The Owren PT method specifies one volume of anticoagulated plasma and 20 volumes of reagent. The greater sample dilution of the Owren method reduces matrix effects. This makes the Owren method more accurate that the Quick method.
Adaptation of a laboratory PT method to needs of smaller laboratories and near-patient assay sites requires the use of blood instead of anticoagulated plasma. According to prior art, the PT analyte is found only in the plasma portion of anticoagulated blood, and not in the cell portion. According to this, depending on the anticoagulation process and the hematocrit, the PT in one volume of anticoagulated plasma is assumed to be about the same as in 1.5 volumes of blood. Thus, according to prior art, the upholding of the assay conditions of the Quick PT method or of the Owren PT method requires that 1.5 volumes of blood be mixed with 2 volumes or 20 volumes of reagent, respectively.
Better specificity for vitamin K dependant coagulation factors and better accuracy are advantages of the Owren PT method, compared to the Quick. In spite of this, prior art designers of near-patient PT methods have been more influenced by the teachings of Quick than of Owren. In addition, most near-patient Quick PT method designs have clearly violated the Quick PT assay conditions by mixing blood and dried PT reagent. This has considerably reduced technical problems, but at the risk of further reduced accuracy. Prior art and inventive aspects of near-patient Quick PT methods are described in U.S. Pat. No. 6,402,704 B1 to McMorrow, U.S. Pat. No. 6,103,196 to Yassinzadeh et al, U.S. Pat. No. 5,302,348 to Cusack et al and U.S. Pat. No. 4,849,340 to Oberhardt.
An exception, to general design trends in near-patient PT methods, is the Novi Quick® PT method of November A G, Erlangen, Germany. In spite of its name, the Novi Quick® PT method represents an attempt to adhere to the assay conditions of the Owren PT method. To solve the near-patient problem of precise volumes, the Novi Quick® procedure includes two novel liquid handling devices disclosed in PCT/DE99/00351 and PCT/DE99/01052 to Bertling et al. One of these is a combined glass capillary and hook with which a precise volume of blood can be added to the reagent. The capillary hook is also used to mix the blood and the reagent and, by the procedure of hooking, to determination of the clotting time. The design of the Novi Quick® PT abides to the philosophy of close adherence to accurate laboratory methods. However, in spite of inventive efforts, the requirement of precise volumes has prohibited wide spread use.
The results of a PT determination according to the methods of Quick and Owren are commonly expressed in International Normalized Ratio (INR). The INR of plasma is derived from the quotient of the clotting time divided by the normal clotting time, NCT. To obtain the INR, the quotient is raised to an exponent that is characteristic of the assay procedure. The exponent, together with the NCT, is determined by calibration. The exponent is called the International Sensitivity Index (ISI). Alternatively, PT can be expressed with respect to the PT of normal plasma, herein called PT %. Equations for inter-conversion of PT % and INR; PT %=1/(0.028*INR−0.018) and INR=[(1/PT %)+0.018]/0.028, are given in Lindahl et al. INR calibration of Owren type prothrombin time based on the relationship between PT % and INR utilizing normal plasma samples. Submitted to Thrombosis and Haemostasis. Similar information is found in Gogstad G. The reporting of thrombotest in international normalized ratio (INR). Farmakoterapi 1984; 40: 88-92.
Some of the difficulties encountered in attempts to harmonize the results of PT determinations in blood and PT determinations in anticoagulated plasma are caused by variations in hematocrit. According to prior art, the results are harmonized by use of one or more scaling factors. This gives reasonable results when the hematocrit is in the normal range, but not when the hematocrit is in the extremes.
Hematocrit is the fraction of the blood volume that is made up of blood cells. Hematocrit can be determined by exposing a container with blood to centrifugal forces. The blood cells then form a compact mass at the bottom of the container, the volume of which is measured to determine the hematocrit. Measuring and summing the volume of each individual blood cell is another way to determine hematocrit. There are also optical methods. These are based on the fact that a good majority of blood cells are red blood cells filled with the red colored protein hemoglobin, the light absorption of which can be measured by optical methods to determine the hematocrit. Optical methods to determine hematocrit are convenient and deserve special attention. Background and inventive aspects of optical determination of hematocrit are given in the following publications: U.S. Pat. No. 6,064,474 to Lee et al and U.S. Pat. No. 5,277,181 to Mendelson et al. The first document discloses a method for noninvasive measurement of hematocrit and hemoglobin content of blood using one or more wavelengths, e.g. 815 nm and 915 nm. The wavelengths are selected to give information on hemoglobin concentration and plasma light scatter. The second document also discloses the use of two wavelengths one at approximately 500 nm and the other at approximately 800 nm. The wavelengths are chosen because, at these, the two main forms of hemoglobin, the oxygen depleted and the oxygen saturated, show about the same adsorption of light.
At smaller laboratories and at near-patient assay sites there is a need of accurate wet-chemistry methods to determine an analyte concentration in anticoagulated plasma by performing analysis on the corresponding blood, i.e. there is a need to determine an analyte concentration of anticoagulated plasma without having to prepare the same—only to imagine or postulate its existence and its relevant properties. At near-patient assay sites, the methods need to be practiced in a way that circumvents the requirement of precisely defined volumes of blood and reagent. For good assay reliability, the methods should be such that regular control material, typically control plasmas and control serum with known or determined analyte concentration, can be tested. In addition, the methods should be practiced on calibrated analytical set-ups that are regularly checked by analysis of control samples, and measurement and determination devices used should be periodically maintained, i.e. serviced and checked. There is also a need of methods with which the above is possible. There is a need of measurement and determination devices with which such methods can be reliably practiced, and there is a need of equipment kits for the same. Specifically, all the above is needed in PT determinations used to monitor anticoagulation therapy with vitamin K antagonists.